Figure 4. Arterial and venous-derived endothelial cells populate the dorsal part of venous ISV and contribute to sprouting upon loss of flt1.

(a) Endothelial cell tracking in Tg(kdrl:nlsKikGR)^hsc7 embryos. Photo-converted PCV-derived venous endothelial cells express red-kikGR. (b–f) Endothelial cell tracing during 30–60 hpf, showed that PCV-derived endothelium, indicated in red & labelled a,b,c,d,e, migrated along the ISV, from ventral to dorsal up to the most dorsal part of vISVs (cell labelled b). Pre-existing arterial endothelial cells in ISV, in green and labelled 1,2,3,4, shows artery-derived ECs in the dorsal part of vISVs (cell labelled 2) adjacent of PCV-derived EC (cell labelled b,a). (g) Schematic representation of the scenario imaged in b–f. (h) Left panel: Identity analysis of endothelial cells in the dorsal part of vISV revealed three different scenarios: (I) mixed, both arterial and venous-derived endothelium were present, (II) only vein-derived endothelium, (III) only artery-derived endothelium. (n=10 experiments & 6ISVs/embryo). Right panel: % of artery and vein-derived endothelium in the mixed population scenario. (i) Schematic representation of the three identity scenarios in dorsal part of vISV. Arterial derived EC in red, venous-derived EC in blue. (j–l) Ectopic sprouting scenarios in flt1 morphants in Tg(flt1^enh:Tdtomato; flt4:mCitrine), n=6 embryos. (j) Ectopic venous sprout devoid of flt1^enh expressing artery-derived EC (arrowhead). (k) Ectopic venous sprout (arrowhead) containing both arterial and venous-derived ECs; the flt1^enh expressing artery-derived EC is juxtaposed to the venous-derived cell at the tip (arrowhead). (l) Ectopic venous sprout only containing flt1^enh expressing artery-derived ECs; flt1^enh (red) and flt4 (green) were expressed by the same cell which appears in yellow (arrowhead). (m–o) Schematic representation of the three ectopic venous sprouting scenarios. (p) Ectopic sprouting upon loss of flt1 in Tg(flt1^enh:Tdtomato; flt4:mCitrine) (representative of 5 embryos). Flt1^enh positive sprouts (pink arrowhead) and sprouts devoid of flt1^enh (white arrowhead). MO, flt1 morpholino, 1ng. aISV, arterial intersegmental vessel; DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; PCV, posterior cardinal vein; vISV, venous intersegmental vessel. Scale bar, 20 μm in j–l, 10 μm in b–f,p.